Expression of recombinant proteinase k &lt;I&gt; from tritirachium album &lt;/I&gt;in yeast

ABSTRACT

The invention concerns a method for the expression of a gene coding for a soluble proteinase K in yeast e.g. in  Pichia pastoris  with subsequent secretion into the culture medium. In addition a method for purifying the heterologously expressed and secreted proteinase K is described.

[0001] The present invention concerns a method for the production ofrecombinant proteinase K in a soluble and active form in economicallyrelevant amounts.

[0002] Proteinase K (E.C. 3.4.21.64, also known as endopeptidase K) isan extracellular endopeptidase which is synthesized by the fungusTritirachium album Limber. It is a member of the class of serineproteases with the typical catalytic triad Asp³⁹-His⁶⁹-Ser²²⁴ (Jany, K.D. et al. (1986) FEBS Letters Vol. 199(2), 139-144). Since the sequenceof the polypeptide chain of 279 amino acids in length (Gunkel, F. A. andGassen, H. G. (1989) Eur. J. Biochem. Vol. 179(1), 185-194) and thethree dimensional structure (Betzel, C. et al. (1988) Eur. J. Biochem.Vol. 178(1), 155-71) has a high degree of homology to bacterialsubtilisins, proteinase K is classified as a member of the subtilisinfamily (Pahler, A. et al. (1984) EMBO J. Vol. 3(6), 1311-1314; Jany, K.D. and Mayer, B. (1985), Biol. Chem. Hoppe-Seyler, Vol. 366(5),485-492). Proteinase K was named on the basis of its ability tohydrolyse native keratin and thus allows the fungus to grow on keratinas the only source of carbon and nitrogen (Ebeling, W. et al. (1974)Eur. J. Biochem. Vol. 47(1), 91-97). Proteinase K has a specificactivity of more than 30 U/mg and is thus one of the most active of theknown endopeptidases (Betzel, C. et al. (1986) FEBS Lett. Vol. 197(1-2),105-110) and unspecifically hydrolyses native and denatured proteins.

[0003] Proteinase K from Tritirachium album Limber is translated in itsnatural host as a preproprotein. The sequence of the cDNA of the genewhich codes for proteinase K was decoded in 1989 by Gunkel, F. A. andGassen, H. G. (1989) Eur. J. Biochem. Vol. 179(1), 185-194. According tothis the gene for prepro-proteinase K is composed of two exons and codesfor a signal sequence of 15 amino acids in length, a prosequence of 90amino acids in length and a mature proteinase K of 279 amino acids inlength. A 63 bp intron is located in the region of the prosequence. Theprepeptide is cleaved off during translocation into the endoplasmaticreticulum (ER). At present very little is known about the subsequentprocessing to form mature proteinase K with cleavage of the propeptide.

[0004] Consequently mature proteinase K consists of 279 amino acids. Thecompact structure is stabilized by two disulfide bridges and two boundcalcium ions. This explains why proteinase K compared to othersubtilisins has a considerably higher stability towards extreme pHvalues, high temperatures, chaotropic substances and detergents(Dolashka, P. et al. (1992) Int. J. Pept. Protein. Res. Vol. 40(5),465-471). Proteinase K is characterized by a high thermostability (up to65° C., Bajorath et al. (1988), Eur. J. Biochem. Vol. 176, 441-447) anda wide pH range (pH 7.5-12.0, Ebeling, W. et al. (1974) Eur. J. Biochem.Vol. 47(1), 91-97). Its activity is increased in the presence ofdenaturing substances such as urea or SDS (Hilz, H. et al. (1975) J.Biochem. Vol. 56(1), 103-108; Jany, K. D. and Mayer, B. (1985) Biol.Chem. Hoppe-Seyler, Vol. 366(5), 485-492).

[0005] The above-mentioned properties make proteinase K of particularinterest for biotechnological applications in which an unspecificprotein degradation is required. Special examples are nucleic acidisolation (DNA or RNA) from crude extracts and sample preparation in DNAanalysis (Goldenberger, D. et al. (1995) PCR Methods Appl. Vol. 4(6),368-370; U.S. Pat. No. 5,187,083; U.S. Pat. No. 5,346,999). Otherapplications are in the field of protein analysis such as structureelucidation.

[0006] Proteinase K is obtained commercially in large amounts byfermentation of the fungus Tritirachium album Limber (e.g. CBS 348.55,Merck strain No. 2429 or the strain ATCC 22563). However, in thisprocess the production of proteinase K is suppressed by glucose or freeamino acids. Since protein-containing media also induce the expressionof proteases, it is necessary to use proteins such as BSA, milk powderor soybean flour as the only nitrogen source. The secretion of theprotease starts as soon as the stationary phase of growth is reached(Ebeling, W. et al. (1974) Eur. J. Biochem. Vol. 47(1), 91-97).

[0007] Since Tritirachium album Limber is consequently unsuitable forfermentation on a large scale and moreover is difficult to geneticallymanipulate, various attempts have been made to overexpress recombinantproteinase K in other host cells. However, no significant activity wasdetected in these experiments due to lack of expression, formation ofinactive inclusion bodies or problems with the renaturation (Gunkel, F.A. and Gassen, H. G. (1989) Eur. J. Biochem. Vol. 179(1), 185-194;Samal, B. B. et al. (1996) Adv. Exp. Med. Biol. Vol. 379, 95-104).

[0008]Tritirachium album Limber is a slowly growing fungus which onlysecretes small amounts of proteases into the medium. It has thedisadvantage of a slower cell cycle compared to yeast and the loweroptical density that can be achieved in a fermenter. In addition it isknown that T. album also produces other proteases apart from proteinaseK which can contaminate the preparation (Samal, B. B. et al. (1991)Enzyme Microb. Technol. Vol. 13, 66-70).

[0009] Although in principle it is possible to express proteinase K inE. coli, it is not expressed in a soluble form but in so-calledinclusion bodies from which the enzyme has to be subsequentlysolubilized and renatured by certain measures. A disadvantage of thismethod is that a lot of protein is lost during the solubilization andrenaturing.

[0010] Hence the object of the present invention is to provide a methodfor producing recombinant proteinase K in economically relevant amounts.

[0011] It has surprisingly turned out that it is possible to express andsecrete recombinant proteinase K as a zymogenic precursor in a solubleform in yeast which is autocatalytically activated to form activeproteinase K. Another subject matter of the invention is thepurification of active proteinase K from the medium supernatant.

[0012] Hence the present invention concerns a method for producingrecombinant proteinase K comprising the steps:

[0013] a) transformation of a host cell with a vector containing a DNAcoding for the zymogenic precursor of proteinase K which is fusedupstream of the coding sequence with a sequence in the reading framewhich codes for a signal peptide and is under the control of a suitablepromoter for the host cell,

[0014] b) expression of the zymogenic precursor of proteinase K

[0015] c) secretion and autocatalytic activation of proteinase K

[0016] d) isolation and purification of proteinase K,

[0017] characterized in that the host cell is a yeast cell and theprotein is secreted in a soluble form by this expression host.

[0018] In a special embodiment of the method according to the inventionthe host cell is selected from the following group: Pichia species,Hansenula species such as Hansenula polymorpha, Saccharomyces species,Schizosaccharomyces species, Yarrowia species such as Yarrowialipolytica, Kluyveromyces species and Aspergillus species. It isparticularly preferred according to the invention when Pichia pastorisis used as the host cell.

[0019] Furthermore it has proven to be advantageous for the methodaccording to the invention when the host cell is transformed with a DNAcoding for the zymogenic precursor and the proteinase K isautocatalytically activated at a later time during or immediately aftersecretion into the culture medium.

[0020] When using Pichia pastoris as a host cell, the gene coding forthe zymogenic precursor of proteinase K is preferably cloned into thefollowing vectors: pPICZ, pPICZα, pGAPZ, pGAPZα, pPICZαA and pPIC9K. Inthis case the vectors: pPICZαA and pPIC9K are particularly preferred.According to the invention the vector pPICZαA is particularly preferred.The above-mentioned vectors are commercially available (Invitrogen).

[0021] In addition in the inventive method for producing recombinantproteinase K it is preferred that the expression of proteinase K or thezymogenic precursor of proteinase K is induced by methanol (pPICvectors). Another method is to induce the expression by glyceraldehydephosphate (pGAP vectors).

[0022] In the inventive method for producing recombinant proteinase Kthe secretion of the protein is preferably initiated by the N-terminalfusion of the signal peptide of the α-factor from Saccharomycescerevisiae. This for example means that the above-mentioned α-labelledvectors have the nucleotide sequence for the signal peptide of theα-factor from Saccharomyces cerevisiae. A fusion protein consisting ofthe signal peptide at the N-terminus and the target protein is thenproduced during translation. Another possible signal peptide would bethe natural signal sequence for proteinase K.

[0023] Furthermore it has proven to be particularly advantageous for theproduction of recombinant proteinase K, to transform the host cellPichia pastoris with the expression vectors pPICZαA and pPIC9K whichcontain a DNA coding for the zymogenic precursor and that the gene isunder the control of the AOX1 promoter and optionally of the AOX1terminator.

[0024] The present invention also concerns a vector containing a DNAcoding for the zymogenic precursor of proteinase K which is fusedupstream of the coding sequence with a sequence in the reading framewhich codes for a suitable signal peptide and wherein the coding gene isunder the control of a suitable promoter and optionally terminator forthe host cell and wherein this vector is suitable for the transformationof this host cell. According to the invention the host cell is a yeast.

[0025] Hence the invention also concerns a recombinant vector whichcontains one or more copies of the recombinant DNA defined above. Thevector is preferably a plasmid which has a strong promoter for the hostcell and a suitable signal peptide for the host cell for secretingproteins. Moreover it is also possible to fuse the native signal peptideof prepro-proteinase K to the N-terminus of the propeptide as shown inSEQ ID NO.: 21 (signal sequence 1-15 (15 amino acids); prosequence16-104 (90 amino acids); sequence of the mature proteinase K 106-384(279 amino acids)). Methods are used to produce the expression vectorwhich are familiar to a person skilled in the art and are described forexample in Sambrook et al. (1989).

[0026] Another subject matter of the present invention is a host celltransformed with one of the vectors listed above where the host cell isa yeast. The host cell is preferably selected from the following group:Pichia species, Hansenula species such as Hansenula polymorpha,Saccharomyces species, Schizosaccharomyces species, Yarrowia speciessuch as Yarrowia lipolytica, Kluyveromyces species and Aspergillusspecies. Pichia pastoris is particularly preferred as the host cell. Inparticular it is preferred when several vectors (each with one copy ofthe ppK gene) are integrated into the genome.

[0027] In addition the present invention concerns a method for purifyingproteinase K. In order to purify the protease the yeast cells areremoved in a first step by microfiltration or centrifugation. Theresulting clear solution contains the protease. This is followed by arebuffering by means of ultrafiltration in order to bind the product toa cation exchanger such as SP-Sepharose or SP-Sephadex (Pharmacia) orSP-Toyopearl (Tosoh Corporation). After the elution it is againrebuffered by means of ultrafiltration and bound to an anion exchangersuch as DEAE-Sepharose or Q-Sepharose (Pharmacia) or DEAE-Fraktogel(Merck). After another elution the pure protease is transferred by meansof ultrafiltration into a stable buffer system (Protein Purification,Principles and Practice, Robert K. Scopes, Springer Verlag, 1982).However, a person skilled in the art can use other methods ofpurification which are part of the prior art.

[0028] The method according to the invention surprisingly enables thepreparation of recombinant proteinase K in which the enzyme is producedby a heterologous host cell in a soluble and active form. The expressionof proteinase K with subsequent secretion of the enzyme into the culturemedium is of particular advantage since it prevents proteinase K fromdeveloping a strongly toxic effect in the cytosol of the host cell.Furthermore this ensures the correct formation of the two disulfidebridges which could not readily occur in the reducing environment of thecytosol. Hence an important advantage of the method according to theinvention is that it provides an approach for the soluble and activeproduction of a recombinant proteinase K. It is very surprising andinexplicable that the surface proteins of the host cells according tothe invention are not hydrolysed by a secreted proteinase K. Such anexpected hydrolysis of the surface proteins by proteinase K wouldinterfere with the life cycle of the host cell.

[0029] A proteinase K is obtained by the method according to theinvention which is homogeneous and hence particularly suitable foranalytical and diagnostic applications. The zymogenic precursor ofproteinase K according to the invention can optionally containadditional N-terminal modifications and in particular sequences whichfacilitate purification of the target protein (affinity tags). Inaddition the zymogenic precursor can contain sequences which increasethe efficiency of translation, which increase the folding efficiencyand/or also sequences which result in a secretion of the target proteininto the culture medium (natural presequence and other signal peptides).

[0030] Proteinase K in the sense of the invention means the sequenceaccording to Gassen et al. (1989) shown in SEQ ID NO:1 as well as othervariants of proteinase K from Tritirachium album Limber like the aminoacid sequence disclosed by Ch. Betzel et al. (Biochemistry 40 (2001),3080-3088) and in particular proteinase T (Samal, B. B. et al. (1989)Gene Vol. 85(2), 329-333; Samal, B. B. et al. (1996) Adv. Exp. Med.Biol. Vol. 379, 95-104) and proteinase R (Samal, B. B. et al. (1990)Mol. Microbiol. Vol. 4(10), 1789-1792; U.S. Pat. No. 5,278,062) and inaddition variants produced by recombinant means (as described forexample in WO 96/28556). SEQ ID NO:1 comprises a prosequence (1-90; 90amino acids) and the sequence of the mature proteinase K (91-368; 279amino acids). The proteinase K amino acid sequence described by Betzelet al. (Biochemistry 40 (2001), 3080-3088) has in particular aspartateinstead of a serine residue at position 207 of the active protease.

[0031] Pro-proteinase K in the sense of the invention means inparticular a proteinase K whose N-terminus is linked to its prosequenceaccording to SEQ ID NO: 1. In the case of subtilisin E which is closelyrelated to proteinase K and variants thereof, the prosequence has animportant influence on the folding and formation of active protease(Ikemura, H. et al. (1987) J. Biol. Chem. Vol. 262(16), 7859-7864). Inparticular it is postulated that the prosequence acts as anintramolecular chaperone (Inouye, M. (1991) Enzyme Vol. 45, 314-321).After the folding it is processed to form the mature subtilisin proteaseby autocatalytically cleaving the propeptide (Ikemura, H. and Inouye, M.(1988) J. Biol. Chem. Vol. 263(26), 12959-12963). This process occurs inthe case of subtilisin E (Samal, B. B. et al. (1989) Gene Vol. 85(2),329-333; Volkov, A. and Jordan, F. (1996) J. Mol. Biol. Vol. 262,595-599), subtilisin BPN′ (Eder, J. et al. (1993) Biochemistry Vol. 32,18-26), papain (Vernet, T. et al. (1991) J. Biol. Chem. Vol. 266(32),21451-21457) and thermolysin (Marie-Claire, C. (1998) J. Biol. Chem.Vol. 273(10), 5697-5701).

[0032] Only certain core regions of the prosequence which are usuallyhydrophobic appear to be necessary for the chaperone function since awide range of mutations have no influence on the activity (Kobayashi, T.and Inouye, M. (1992) J. Mol. Biol. Vol. 226, 931-933). In addition itis known that propeptides can be interchanged between various subtilisinvariants. Thus for example subtilisin BPN′ also recognizes theprosequence of subtilisin E (Hu, Z. et al. (1996) J. Biol. Chem. Vol.271(7), 3375-3384).

[0033] Hence the present invention concerns the prosequence according toSEQ ID NO:1 of 90 amino acids in length as well as other variants whichfacilitate folding. It also concerns a propeptide which is addedexogenously for the folding of mature proteinase K and has the functionsdescribed above.

[0034] Hence an important advantage of the method according to theinvention is that the recombinant proteinase K is secreted by anexpression host into the culture medium in a soluble and active form.Moreover the expression host used in the method according to theinvention is not damaged or otherwise impaired by the very active andunspecific protease i.e. in particular it continues to grow withoutproblems and an increased cell lysis is not observed. Furthermore theexpression host according to the invention is easier to handle comparedto Tritirachium album and is characterized by higher growth rates.

DESCRIPTION OF THE FIGURES

[0035]FIG. 1

[0036] Expression plasmid pPICPK-1. A sequence coding for the zymogenicproform of proteinase K cloned into the starting vector pPICZαA(Invitrogen).

[0037]FIG. 2

[0038] Expression plasmid pPICPK-2. A sequence coding for the zymogenicproform of proteinase K cloned into the starting vector pPIC9K(Invitrogen).

EXAMPLES Example 1 Gene Synthesis

[0039] The gene for mature proteinase K from Tritirachium album Limberwithout a signal sequence and without an intron was generated by meansof gene synthesis. The sequence (without a native signal peptide) ofGunkel and Gassen, 1989 of 368 amino acids in length was used as atemplate. A codon-optimized nucleic acid sequence for expression in E.coli as well as yeast was obtained by retranslating the amino acidsequence. The amino acid sequence is shown in SEQ ID NO:1 and thenucleotide sequence is shown in SEQ ID NO:2.

[0040] For the gene synthesis the gene was divided into 18 fragments ofsense and reverse, complementary counterstrand oligonucleotides inalternating sequence (SEQ ID NO: 3-20). An at least 15 bp region wasattached to the 5′ end and to the 3′ end which in each case overlappedthe neighbouring oligonucleotides. Recognition sites for restrictionendonucleases were attached to the 5′ and 3′ ends of the synthetic geneoutside the coding region for subsequent cloning into expressionvectors. The oligonucleotide shown in SEQ ID NO: 3 which contains anEcoRI cleavage site was used as a 5′ primer for cloning thepro-proteinase K gene. SEQ ID NO: 20 shows the 3′ primer containing aHindIII cleavage site. The 3′ primer contains an additional stop codonafter the natural stop codon to ensure termination of the translation.

[0041] The oligonucleotides were linked together by means of a PCRreaction and the resulting gene was amplified. For this the gene wasfirstly divided into three fragments of 6 oligonucleotides each and thethree fragments were linked together in a second PCR cycle.

[0042] Fragment 1 is composed of the oligonucleotides shown in SEQ IDNO: 3-8, fragment 2 is composed of the oligonucleotides shown in SEQ IDNO: 9-14 and fragment 3 is composed of the oligonucleotides shown in SEQID NO: 15-20.

[0043] The following PCR parameters were applied

[0044] PCR Reaction 1 (Generation of Three Fragments) 5 min 5° C. hotstart 2 min 95° C. 2 min 42° C. 30 cycles 1.5 min   72° C. {closeoversize brace} 7 min 72° C. final extension

[0045] PCR Reaction 2 (Linkage of the Fragments to Form the Total Gene)5 min 95° C. hot start 1.5 min   95° C. 2 min 48° C. {close oversizebrace} 6 cycles (without terminal primers) 2 min 72° C.

[0046] Addition of Terminal Primers 1.5 min 95° C. 1.5 min 60° C. {closeoversize brace} 25 cycles (with terminal primers)   2 min 72° C.   7 min72° C. final extension

Example 2 Cloning of the Synthetic Proteinase K Fragment from the GeneSynthesis

[0047] The PCR mixture was applied to an agarose gel and the ca. 1130 bpPCR fragment was isolated from the agarose gel (Geneclean II Kit fromBio 101, Inc. California USA). The fragment was cleaved for 1 hour at37° C. with the EcoRI and HindIII restriction endonucleases (RocheDiagnostics GmbH, Germany). At the same time the pUC18 plasmid (RocheDiagnostics GmbH, Germany) was cleaved for 1 hour at 37° C. with theEcoRI and HindIII restriction endonucleases, the mixture was separatedby agarose gel electrophoresis and the 2635 bp vector fragment wasisolated. Subsequently the PCR fragment and the vector fragment wereligated together using T4 DNA ligase. For this 1 μl (20 ng) vectorfragment and 3 μl (100 ng) PCR fragment, 1 μl 10×ligase buffer (Maniatiset al., 1989, B.27), 1 μl T4 DNA ligase, 4 μl sterile redistilled H₂Owere pipetted, carefully mixed and incubated overnight at 16° C.

[0048] The cloned gene was examined by restriction analysis and bymultiple sequencing of both strands.

Example 3 Vector Construction

[0049] The synthetic gene has to be firstly isolated again from the pUCplasmid. For this purpose 1 μg plasmid DNA was firstly incubated withthe restriction endonuclease HindIII (Roche Diagnostics GmbH) accordingto the manufacturer's instructions and subsequently the restrictionendonuclease was inactivated by heating to 65° C. for 20 min. Afterwardsthe resulting DNA overhangs were filled in with Klenow polymeraseaccording to the manufacturer's instructions (Roche Diagnostics GmbH)and the Klenow polymerase was then inactivated by incubating at 75° C.for 10 min. Finally the vector fragment which was now linearized of theabove-mentioned pUC plasmid was cleaved with the restrictionendonuclease EcoRI (Roche Diagnostics GmbH) according to themanufacturer's instructions, the reaction mixture was applied to a 1%agarose gel and the fragments were separated according to size byapplying a current (100 V/150 mA). The ca. 1120 bp fragment containingthe gene for pro-proteinase K (ppk gene) was isolated from the agarosegel (QIAquick Gel Extraction Kit/Qiagen).

[0050] The vector pPICZαA (Invitrogen) was cleaved with the restrictionendonuclease Asp718I (Roche Diagnostics GmbH) according to themanufacturer's instructions and the restriction endonuclease wasinactivated by heating the incubation mixture to 65° C. for 20 min.Afterwards the resulting DNA overhangs were filled in with Klenowpolymerase according to the manufacturer's instructions (RocheDiagnostics GmbH) and the Klenow polymerase was then inactivated byincubating at 75° C. for 10 min. Finally the vector fragment which wasnow linearized of pPICZαA was cleaved with the restriction endonucleaseEcoRI (Roche Diagnostics GmbH) according to the manufacturer'sinstructions, the reaction mixture was applied to a 1% agarose gel andthe fragments were separated according to size by applying a current(100 V/150 mA). The ca. 3550 bp vector fragment was isolated from theagarose gel (QIAquick Gel Extraction Kit/Qiagen).

[0051] The fragments obtained in this manner were ligated together bystandard methods (Sambrook et al. 1989). In this vector the ppk gene isunder the control of the AOX-1 promoter (promoter for alcohol oxidase 1from Pichia pastoris, inducible with methanol) and is cloned using thiscloning strategy in the correct reading frame behind the signal peptideof the α-factor from Saccharomyces cerevisiae. The gene fragmentinserted in this manner was then examined for an error free sequence bymeans of restriction analysis and sequencing. The resulting expressionvector which contains the ppk gene which codes for pro-proteinase K wasnamed pPICPK-1 (see FIG. 1).

[0052] Subsequently the ppk gene was also cloned into pPIC9K(Invitrogen). For this purpose the vector pPICPK-1 was cleaved accordingto the manufacturer's instructions with the restriction endonucleasesPmeI and NotI (Roche Diagnostics GmbH), the fragments from therestriction mixture were separated according to size in a 1% agarose geland the ca. 1960 bp fragment containing the 3′ part of the AOX1-promoterregion, the sequence for the signal peptide of the α-factor and the ppkgene was isolated from the gel (QIAquick Gel Extraction Kit/Qiagen). Atthe same time the vector pPIC9K was cleaved with the restrictionendonucleases PmeI and NotI (Roche Diagnostics GmbH) according to themanufacturer's instructions, the fragments from the restriction mixturewere separated according to size in a 1% agarose gel and the ca. 8450 bpvector fragment was isolated from the gel (QIAquick Gel ExtractionKit/Qiagen).

[0053] Subsequently the fragments obtained in this manner were ligatedtogether by standard methods (Sambrook et al. 1989). In this vector theppk gene is also under the control of the AOX1-promoter (promoter forthe alcohol oxidase 1 from Pichia pastoris, inducible with methanol).The vector pPIC9K differs from the vector pPICZαA by the selectionmarker and by three possibilities known to a person skilled in the artfor integrating it into the Pichia genome depending on the vectorlinearization before transformation whereas the integration of pPICZαAinto the AOX1-locus is fixed. The inserted gene fragment was thenexamined for an error-free sequence by means of restriction analysis andsequencing.

[0054] The resulting expression vector which contains the ppk gene whichcodes for pro-proteinase K was named pPICPK-2 (see FIG. 2).

Example 4 Transformation of pPICPK-1 in Pichia pastoris

[0055] In order to transform pPICPK-1 in Pichia pastoris X-33 withsubsequent integration into the genome, the vector was firstlylinearized with PmeI (Roche Diagnostics GmbH). The transformation wascarried out by means of electroporation using a Gene Pulser II (Biorad).

[0056] For this purpose 5 ml YPD medium (according to the Invitrogencatalogue) was inoculated with a colony of Pichia pastoris wild-typestrain and incubated overnight at 30° C. while shaking. The overnightculture was subsequently reinoculated 1:2000 in 200 ml fresh YPD medium(according to the Invitrogen catalogue) and incubated overnight at 30°C. while shaking until the OD₆₀₀ reached 1.3-1.5. The cells werecentrifuged (1500×g/5 minutes) and the pellet was resuspended in 200 mlice-cold sterile water (0° C.). The cells were again centrifuged(1500×g/5 minutes) and resuspended in 100 ml ice-cold sterile water (0°C.). The cells were again centrifuged and resuspended in 10 ml ice-cold(0° C.) 1 M sorbitol (ICN). The cells were again centrifuged andresuspended in 0.5 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cellsobtained in this manner were kept on ice and used immediately fortransformation.

[0057] About 1 μg linearized pPICPK-1 vector DNA was added to 80 μl ofthe cells and the entire mixture was transferred to an ice-cold (0° C.)electroporation cuvette and incubated for a further 5 minutes on ice.Subsequently the cuvette was transferred to a Gene Pulser II (Biorad)and the transformation was carried out at 1 kV, 1 kΩ and 25 μF. Afterelectroporation 1 ml 1 M sorbitol (ICN) was added to the mixture andsubsequently 100-150 μl was plated out on a YPDS agar plate (accordingto the Invitrogen catalogue) containing 100 μg/ml Zeocin® (Invitrogen).The plates were subsequently incubated for 2-4 days at 30° C.

[0058] Minimal dextrose grid plates were inoculated with the clones andthe clones were analysed further.

[0059] Clones that had grown were picked, resuspended in 20 μl sterilewater and lysed with 17.5 U lyticase (Roche Diagnostics GmbH) (1 hour,37° C.) and examined directly by means of PCR for the correctintegration of the ppk expression cassette.

[0060] Clones which had integrated the complete expression cassetteduring transformation into the genome were then used in expressionexperiments.

Example 5 Transformation of pPICPK-2 in Pichia pastoris

[0061] In order to transform pICPK-2 in Pichia pastoris GS115 withsubsequent integration into the genome, the vector was firstlylinearized for variant I with PmeI (Roche Diagnostics GmbH) to integrateit into the AOXI-locus and linearized with SalI (Roche Diagnostics GmbH)for variant II to integrate it into the His4 locus. The transformationwas carried out by means of electroporation using a Gene Pulser(Biorad).

[0062] For this purpose 5 ml YPD medium (according to the Invitrogencatalogue) was inoculated with a colony of Pichia pastoris GS115wild-type strain and incubated overnight at 30° C. while shaking. Theovernight culture was subsequently reinoculated 1:2000 in 200 ml freshYPD medium (according to the Invitrogen catalogue) and incubatedovernight at 30° C. while shaking until the OD₆₀₀ reached 1.3-1.5. Thecells were centrifuged (1500×g/5 minutes) and the pellet was resuspendedin 200 ml ice-cold sterile water (0° C.). The cells were againcentrifuged (1500×g/5 minutes) and resuspended in 100 ml ice-coldsterile water (0° C.). The cells were again centrifuged and resuspendedin 10 ml ice-cold (0° C.) 1 M sorbitol (ICN). The cells were againcentrifuged and resuspended in 0.5 ml ice-cold (0° C.) 1 M sorbitol(ICN). The cells obtained in this manner were kept on ice and usedimmediately for transformation.

[0063] About 1 μg linearized pPICPK-2 vector DNA was added to 80 μl ofthe cells and the entire mixture was transferred to an ice-cold (0° C.)electroporation cuvette and incubated for a further 5 minutes on ice.Subsequently the cuvette was transferred to a Gene Pulser II (Biorad)and the transformation was carried out at 1 kV, 1 kΩ and 25 μF. Afterelectroporation 1 ml 1 M sorbitol (ICN) was added to the mixture andsubsequently 100-150 μl was plated out on a MM agar plate (minimalmedium according to the Invitrogen catalogue) without histidine. Theplates were subsequently incubated for 2-4 days at 30° C. Clones ofPichia pastoris GS115 which have a defective His4 gene caused bymutation (histidinol dehydrogenase) can only grow on these plates whenthey have integrated the vector pPICPK-2 which has a functional His4gene as an insert and can hence compensate the deficiency in histidinebiosynthesis.

[0064] Minimal dextrose grid plates were inoculated with the clones andthe clones were analysed further. Clones that had grown were picked,resuspended in 20 μl sterile water and lysed with 17.5 U lyticase (RocheDiagnostics GmbH) (1 hour, 37° C.) and examined directly by means of PCRfor the correct integration of the ppk expression cassette.

[0065] Clones which had integrated the complete expression cassetteduring transformation into the genome were then used in expressionexperiments.

Example 6 Expression of Proteinase K

[0066] 10 ml BMGY medium (according to the Invitrogen catalogue) wasinoculated with positive clones and incubated overnight at 30° C. whileshaking. Subsequently the optical density at 600 nm was determined and10 ml BMMY medium (according to the Invitrogen catalogue) was inoculatedin such a manner that an OD₆₀₀ of 1 resulted. The BMMY medium (accordingto the Invitrogen catalogue) contains methanol (Mallinckrodt Baker B.V)which induces the expression of proteinase K via the AOX1 promoter.

[0067] The shaking flask was incubated at 30° C. while shaking, sampleswere taken every 24 hours, the OD₆₀₀ was determined, an activity testwas carried out for expression of proteinase K and each time 0.5%methanol (Mallinckrodt Baker B.V) was refed for further induction. Theexpression experiments ran for 168 hours.

Example 7 Activity Test for Secreted Recombinant Proteinase K

[0068] For the activity test for recombinant proteinase K one requiresCaCl₂×2H₂O (Merck ID-No. 102382), DMSO (Merck, ID-No. 109678), thesubstrate Suc-Ala-Ala-Pro-Phe-pNA (Roche Diagnostics ID-No. 0716766) andTris base (Roche Diagnostics ID-No. 0153265).

[0069] The composition of the solutions was as follows:

[0070] Solution 1: 50 mmol/l Tris-Base; 10 mmol/l CaCl₂ pH 8.2

[0071] Solution 2: 125 mg Suc-Ala-Ala-Pro-Phe-pNA dissolved in 1 ml DMSO

[0072] The cells were centrifuged (5 min 10000 rpm Eppendorf benchcentrifuge) and the supernatant was diluted 1:500 in solution 1.

[0073] 2 ml of solution 1 was pipetted into a cuvette and 0.02 ml ofsolution 2 was added. Both solutions were mixed and incubated at areaction temperature of 25° C. The reaction was started by adding 0.05ml of the diluted supernatant as stated above and remixing, the changein absorbance at 405 nm was measured and the ΔA/min in the linear regionwas measured. The following formula was then used for the calculation:${activity} = {\frac{2.07}{\varepsilon \times 1 \times 0.05}\Delta \quad A\text{/}{\min \left\lbrack {U\text{/}{ml}\quad {sample}\quad {solution}} \right\rbrack}}$

[0074] 2.07=sample volume

[0075] ε₄₀₅=10.4 [mmol⁻¹×1×cm⁻¹]

[0076] l=path length of the cuvette

[0077] 0.05=volume of the added sample

1 21 1 369 PRT Tritirachium album Limber 1 Ala Pro Ala Val Glu Gln ArgSer Glu Ala Ala Pro Leu Ile Glu Ala 1 5 10 15 Arg Gly Glu Met Val AlaAsn Lys Tyr Ile Val Lys Phe Lys Glu Gly 20 25 30 Ser Ala Leu Ser Ala LeuAsp Ala Ala Met Glu Lys Ile Ser Gly Lys 35 40 45 Pro Asp His Val Tyr LysAsn Val Phe Ser Gly Phe Ala Ala Thr Leu 50 55 60 Asp Glu Asn Met Val ArgVal Leu Arg Ala His Pro Asp Val Glu Tyr 65 70 75 80 Ile Glu Gln Asp AlaVal Val Thr Ile Asn Ala Ala Gln Thr Asn Ala 85 90 95 Pro Trp Gly Leu AlaArg Ile Ser Ser Thr Ser Pro Gly Thr Ser Thr 100 105 110 Tyr Tyr Tyr AspGlu Ser Ala Gly Gln Gly Ser Cys Val Tyr Val Ile 115 120 125 Asp Thr GlyIle Glu Ala Ser His Pro Glu Phe Glu Gly Arg Ala Gln 130 135 140 Met ValLys Thr Tyr Tyr Tyr Ser Ser Arg Asp Gly Asn Gly His Gly 145 150 155 160Thr His Cys Ala Gly Thr Val Gly Ser Arg Thr Tyr Gly Val Ala Lys 165 170175 Lys Thr Gln Leu Phe Gly Val Lys Val Leu Asp Asp Asn Gly Ser Gly 180185 190 Gln Tyr Ser Thr Ile Ile Ala Gly Met Asp Phe Val Ala Ser Asp Lys195 200 205 Asn Asn Arg Asn Cys Pro Lys Gly Val Val Ala Ser Leu Ser LeuGly 210 215 220 Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala Ala Ala Arg LeuGln Ser 225 230 235 240 Ser Gly Val Met Val Ala Val Ala Ala Gly Asn AsnAsn Ala Asp Ala 245 250 255 Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser ValCys Thr Val Gly Ala 260 265 270 Ser Asp Arg Tyr Asp Arg Arg Ser Ser PheSer Asn Tyr Gly Ser Val 275 280 285 Leu Asp Ile Phe Gly Pro Gly Thr SerIle Leu Ser Thr Trp Ile Gly 290 295 300 Gly Ser Thr Arg Ser Ile Ser GlyThr Ser Met Ala Thr Pro His Val 305 310 315 320 Ala Gly Leu Ala Ala TyrLeu Met Thr Leu Gly Lys Thr Thr Ala Ala 325 330 335 Ser Ala Cys Arg TyrIle Ala Asp Thr Ala Asn Lys Gly Asp Leu Ser 340 345 350 Asn Ile Pro PheGly Thr Val Asn Leu Leu Ala Tyr Asn Asn Tyr Gln 355 360 365 Ala 2 1124DNA Tritirachium album Limber 2 gaattcgctc ctgccgttga gcagcgctccgaggctgctc ctctgatcga ggcccgcggc 60 gagatggttg ccaacaagta catcgtcaagttcaaggagg gtagcgctct ttccgctctg 120 gatgctgcca tggagaagat ctctggcaagcccgaccacg tctacaagaa cgtcttcagc 180 ggtttcgctg cgaccctgga cgagaacatggttcgggttc tccgcgccca ccccgatgtt 240 gagtacatcg agcaggatgc tgttgtcaccatcaacgctg cgcagaccaa cgctccctgg 300 ggcctggctc gcatctccag caccagccccggtacctcta cctactacta tgacgaatct 360 gccggccaag gctcctgcgt ctacgtgatcgacaccggta tcgaggcatc gcaccccgag 420 ttcgagggtc gtgcccagat ggtcaagacctactactact ccagtcgcga cggtaacggt 480 cacggcaccc actgcgctgg taccgttggctcccgtacct acggtgtcgc caagaagacc 540 cagctgttcg gtgtcaaggt cctggatgacaacggcagtg gccagtactc caccatcatc 600 gccggtatgg acttcgttgc cagcgacaagaacaaccgca actgccccaa aggtgtcgtt 660 gcctccttat ccctgggcgg tggttactcctcctccgtga acagcgccgc tgcccgcctc 720 cagagctctg gtgtcatggt cgccgtcgctgccggtaaca acaacgctga cgcccgcaac 780 tactcccctg cttctgagcc ctcggtctgcaccgtcggtg cttctgaccg ctacgaccgc 840 cgctccagct tctccaacta cggcagcgttttggacatct tcggccctgg taccagcatc 900 ctctccacct ggatcggcgg cagcacccgctccatctctg gtacctccat ggctactccc 960 cacgttgccg gtctcgctgc ctacctcatgactcttggaa agactaccgc cgccagcgct 1020 tgccgataca ttgccgacac cgccaacaagggcgacttaa gcaacattcc cttcggcact 1080 gtcaacttgc ttgcctacaa caactaccaggcttaatgaa gctt 1124 3 79 DNA Artificial Sequence Description ofArtificial Sequence Primer 3 cgcgaattcg ctcctgccgt tgagcagcgc tccgaggctgctcctctgat cgaggcccgc 60 ggcgagatgg ttgccaaca 79 4 80 DNA ArtificialSequence Description of Artificial Sequence Primer 4 atcttctccatggcagcatc cagagcggaa agagcgctac cctccttgaa cttgacgatg 60 tacttgttggcaaccatctc 80 5 80 DNA Artificial Sequence Description of ArtificialSequence Primer 5 tgccatggag aagatctctg gcaagcccga ccacgtctac aagaacgtcttcagcggttt 60 cgctgcgacc ctggacgaga 80 6 64 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 tgctcgatgt actcaacatcggggtgggcg cggagaaccc gaaccatgtt ctcgtccagg 60 gtcg 64 7 65 DNAArtificial Sequence Description of Artificial Sequence Primer 7tgagtacatc gagcaggatg ctgttgtcac catcaacgct gcgcagaccg ctgcgcagac 60caacg 65 8 70 DNA Artificial Sequence Description of Artificial SequencePrimer 8 agtaggtaga ggtaccgggg ctggtgctgg agatgcgagc caggccccagggagcgttgg 60 tctgcgcagc 70 9 80 DNA Artificial Sequence Description ofArtificial Sequence Primer 9 gtacctctac ctactactat gacgaatctg ccggccaaggctcctgcgtc tacgtgatcg 60 acaccggtat cgaggcatcg 80 10 81 DNA ArtificialSequence Description of Artificial Sequence Primer 10 ttaccgtcgcgactggagta gtagtaggtc ttgaccatct gggcacgacc ctcgaactcg 60 gggtgcgatgcctcgatacc g 81 11 78 DNA Artificial Sequence Description of ArtificialSequence Primer 11 ccagtcgcga cggtaacggt cacggcaccc actgcgctggtaccgttggc tcccgtacct 60 acggtgtcgc caagaaga 78 12 73 DNA ArtificialSequence Description of Artificial Sequence Primer 12 atggtggagtactggccact gccgttgtca tccaggacct tgacaccgaa cagctgggtc 60 ttcttggcga cac73 13 81 DNA Artificial Sequence Description of Artificial SequencePrimer 13 ggccagtact ccaccatcat cgccggtatg gacttcgttg ccagcgacaagaacaaccgc 60 aactgcccca aaggtgtcgt t 81 14 81 DNA Artificial SequenceDescription of Artificial Sequence Primer 14 gctctggagg cgggcagcggcgctgttcac ggaggaggag taaccaccgc ccagggataa 60 ggaggcaacg acacctttgg g81 15 82 DNA Artificial Sequence Description of Artificial SequencePrimer 15 gcccgcctcc agagctctgg tgtcatggtc gccgtcgctg ccggtaacaacaacgctgac 60 gcccgcaact actcccctgc tt 82 16 80 DNA Artificial SequenceDescription of Artificial Sequence Primer 16 gttggagaag ctggagcggcggtcgtagcg gtcagaagca ccgacggtgc agaccgaggg 60 ctcagaagca ggggagtagt 8017 83 DNA Artificial Sequence Description of Artificial Sequence Primer17 ctccagcttc tccaactacg gcagcgtttt ggacatcttc ggccctggta ccagcatcct 60ctccacctgg atcggcggca gca 83 18 81 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 18 tcatgaggta ggcagcgaga ccggcaacgtggggagtagc catggaggta ccagagatgg 60 agcgggtgct gccgccgatc c 81 19 81 DNAArtificial Sequence Description of Artificial Sequence Primer 19ctgcctacct catgacctta ggaaagacca ccgccgccag cgcttgccgt tacatcgccg 60acaccgccaa caagggcgac t 81 20 87 DNA Artificial Sequence Description ofArtificial Sequence Primer 20 atataagctt ctattaagcc tggtagttgttgtaggctaa caggttgacg gtgccgaagg 60 gaatgttgct taagtcgccc ttgttgg 87 21384 PRT Tritirachium album Limber 21 Met Arg Leu Ser Val Leu Leu Ser LeuLeu Pro Leu Ala Leu Gly Ala 1 5 10 15 Pro Ala Val Glu Gln Arg Ser GluAla Ala Pro Leu Ile Glu Ala Arg 20 25 30 Gly Glu Met Val Ala Asn Lys TyrIle Val Lys Phe Lys Glu Gly Ser 35 40 45 Ala Leu Ser Ala Leu Asp Ala AlaMet Glu Lys Ile Ser Gly Lys Pro 50 55 60 Asp His Val Tyr Lys Asn Val PheSer Gly Phe Ala Ala Thr Leu Asp 65 70 75 80 Glu Asn Met Val Arg Val LeuArg Ala His Pro Asp Val Glu Tyr Ile 85 90 95 Glu Gln Asp Ala Val Val ThrIle Asn Ala Ala Gln Thr Asn Ala Pro 100 105 110 Trp Gly Leu Ala Arg IleSer Ser Thr Ser Pro Gly Thr Ser Thr Tyr 115 120 125 Tyr Tyr Asp Glu SerAla Gly Gln Gly Ser Cys Val Tyr Val Ile Asp 130 135 140 Thr Gly Ile GluAla Ser His Pro Glu Phe Glu Gly Arg Ala Gln Met 145 150 155 160 Val LysThr Tyr Tyr Tyr Ser Ser Arg Asp Gly Asn Gly His Gly Thr 165 170 175 HisCys Ala Gly Thr Val Gly Ser Arg Thr Tyr Gly Val Ala Lys Lys 180 185 190Thr Gln Leu Phe Gly Val Lys Val Leu Asp Asp Asn Gly Ser Gly Gln 195 200205 Tyr Ser Thr Ile Ile Ala Gly Met Asp Phe Val Ala Ser Asp Lys Asn 210215 220 Asn Arg Asn Cys Pro Lys Gly Val Val Ala Ser Leu Ser Leu Gly Gly225 230 235 240 Gly Tyr Ser Ser Ser Val Asn Ser Ala Ala Ala Arg Leu GlnSer Ser 245 250 255 Gly Val Met Val Ala Val Ala Ala Gly Asn Asn Asn AlaAsp Ala Arg 260 265 270 Asn Tyr Ser Pro Ala Ser Glu Pro Ser Val Cys ThrVal Gly Ala Ser 275 280 285 Asp Arg Tyr Asp Arg Arg Ser Ser Phe Ser AsnTyr Gly Ser Val Leu 290 295 300 Asp Ile Phe Gly Pro Gly Thr Ser Ile LeuSer Thr Trp Ile Gly Gly 305 310 315 320 Ser Thr Arg Ser Ile Ser Gly ThrSer Met Ala Thr Pro His Val Ala 325 330 335 Gly Leu Ala Ala Tyr Leu MetThr Leu Gly Lys Thr Thr Ala Ala Ser 340 345 350 Ala Cys Arg Tyr Ile AlaAsp Thr Ala Asn Lys Gly Asp Leu Ser Asn 355 360 365 Ile Pro Phe Gly ThrVal Asn Leu Leu Ala Tyr Asn Asn Tyr Gln Ala 370 375 380

1. A method for producing recombinant proteinase K comprising the steps:a) transformating a host cell with a vector containing a DNA coding forthe zymogenic precursor of proteinase K which is fused upstream of thecoding sequence with a sequence in the reading frame which codes for asignal peptide and is under the control of a suitable promoter for thehost cell, b) expressing of the zymogenic precursor of proteinase K c)secreting and autocatalytically activating proteinase K wherein the hostcell is a yeast cell and the protein is secreted in a soluble form bythis expression host.
 2. A method as claimed in claim 1, wherein thehost cell is selected from the following group: Pichia sp., Hansenulasp., Saccharomyces sp., Schizosaccharomyces sp., Yarrowia sp.,Kluyveromyces sp. and Aspergillus sp.
 3. A method as claimed in claim 1,wherein the host cell is Pichia pastoris.
 4. A method as claimed inclaim 3, wherein the gene which codes for the zymogenic precursor ofproteinase K is cloned into one of the following vectors pPICZ, pPICZα,pGAPZ, pGAPZα, pPICZαA and pPIC9K.
 5. A method as claimed in claim 3,wherein the host cell is transformed with one of the following vectors:pPICPK-1 and pPICPK-2.
 6. A method as claimed in claim 1, wherein theexpression of the zymogenic precursor of proteinase K is induced bymethanol.
 7. A method as claimed in claim 1, wherein the secretion ofthe protein is initiated by the N-terminal fusion of a signal peptide.8. A method as claimed in claim 1, wherein the secretion of the proteinis initiated by the N-terminal fusion of a signal peptide of theα-factor from Saccharomyces cerevisiae.
 9. A method as claimed in claim1, wherein the host cell Pichia pastoris is transformed with theexpression vector pPICZαA which contains a DNA coding for the zymogenicprecursor of proteinase K and the gene is under the control of the AOX1promoter.
 10. A vector comprising DNA coding for the zymogenic precursorof proteinase K, wherein this DNA is fused upstream of the codingsequence with a sequence in the reading frame which codes for a suitablesignal peptide, and the coding gene is under the control of a suitablepromoter for the host cell and this vector is suitable for thetransformation of yeast.
 11. A vector as claimed in claim 10, whereinthe vector is suitable for the transformation of Pichia pastoris.
 12. Avector as claimed in claim 10, wherein the vector is selected from thefollowing group: pPICZ, pPICZα, pGAPZ, pGAPZα, pPICZαA and pPIC9K.
 13. Avector as claimed in claim 10, wherein the vector is pPICZαA.
 14. Avector as claimed in 10, wherein the vector is pPICPK-1 or pPICPK-2. 15.A host cell transformed with a vector as claimed in one of the claims10-14, wherein the host is a yeast.
 16. A host cell as claimed in claim15, wherein the host cell is selected from the following group: Pichiasp., Hansenula sp., Saccharomyces sp., Schizosaccharomyces sp., Yarrowiasp., Kluyveromyces sp. and Aspergillus sp.
 17. A host cell as claimed inclaim 15, wherein the host cell is Pichia pastoris.